| Hepatocellular carcinoma is the most common primary malignant tumor of the liver,and its mortality has leapt to the second place in cancer induced mortality.Partial hepatectomy and liver transplantation are benefit for patients in early stage of hepatocellular carcinoma,but most patients are diagnosed at the advanced stage.They can only be treated with transcatheter arterial chemoembolization or chemotherapy as a palliative and the treatment effects are not satisfactory.In recent years,immunotherapy of liver cancer has become a research hotspot.Immunotherapy of liver cancer includes cytokine therapy,immunological checkpoint inhibitor therapy,tumor associated antigens(TAAs)targeted therapy and so on.Dendritic cell(DC)vaccine is one of them.DC vaccine is the DCs stimulated with tumor antigen or tumor cell lysate-stimulated DC,which is used as drug to treat cancers via inducing a tumor antigen-specific immune response to kill the tumor cells.A large number of successful applications of DC vaccines in cancer treatment have been reported,which provide preliminary evidences that DC vaccine is safe and effective in the treatment of liver cancer patients..However,an existing problem is that most TAAs are the body's own protein components,and the immune stimulatory signals generated by TAAs are too weak to stimulate the DC to prime innate immune response.Therefore,the addition of stimulatory substances such as appropriate adjuvants to assist stimulating the DC vaccine may lead to better efficacy.In previous studies,we found that curdlan sulfate(CS)was a polysaccharide derivative with immunomodulatory activity.When it was used as an adjuvant to hepatitis B vaccine,it was found that CS could significantly enhance the immunogenicity of HBsAg,promote the activation of antigen-presenting cells and T cells,and increase the level of specific antibodies of hepatitis B antigen,which suggest that CS has potential adjuvant development value.Therefore,in this study,we prepared CSs with different degrees of sulfation and molecular weights,studied their immunomodulatory activities in vitro and screened the CS with the highest adjuvant activity as the DC vaccine adjuvant to treat tumor bearing mice,in order to examine the adjuvant effect of CS.The research content mainly includes:1.The SO3-pyridine reaction system was used to prepare CSs with different degrees of sulfation and molecular weights by controlling the feed ratio and reaction temperature.The prepared samples were characterized by infrared spectrum analyzer,elemental analyzer and multi-angle laser light scattering instrument.2.The proliferation experiment of mouse spleen lymphocytes and the activation experiment of mouse BMDCs were used to determine thein vitro activity of CSs.The CS samples with the highest activity were screened for the in vivo activity study.3.Liver cancer model was established by implanting H22 cells into BALB/c mice.Then the model mice are treated with PBS,unstimulated DC,TCL stimulated DC,CS activated DC,CS+TCL stimulated DC and TCL+LPS stimulated DC vaccines weekly for 4 cycles,respectively.Pharmacodynamic evaluation was performed by comparing tumor volumes and survival rates.4.Immunohistochemistry and realtime fluorescence quantitative PCR were separately used to determine the infiltration of CD8+ T cells as well as the expression levels of MHC ?,MHC? and CD86 and the mRNA expression levels of TNF-??TGF-? and IFN-? in mouse tumor tissues to explore the action mechanisms.5.Dectin-1,TLR2 and TLR4 inhibited DCs were used to study the molecular mechanism of CS in CS-adjuvanted DC vaccine.The results are as follows:1.CSs with different sulfation degrees and molecular weights were successfiully prepared by controlling the proportion of SO3-pyridine and reaction temperature.The CSs are characterized by infrared spectrum analysis,multi-angle laser light scattering analysis and elemental analysis.It was found that the sulfation degree could be controlled by the feeding ratio,and the molecular weights could be controlled by the reaction temperature.2.The immunological activity of CSs was studied.It was found that CSs could promote the proliferation of mouse splenic lymphocytes,induce the maturationmarkers and the expression of costimulatory molecules of dendritic cells.3.The structure-activity relationship of CSs showed that molecular weight had a weak influence on the activity,and the degree of sulfation was strong factor affecting the immunoregulatory activity.The activity was positively correlated with the sulfation degree.4.In vivo study showed that CS adjuvant significantly improved the therapeutic effect of DC vaccine on tumor bearing mice,decreasing the tumor volumes,reducing the tumor weights and prolonging the survival time of the mice.5.In vivo study CS adjuvant added DC vaccine significantly increased the expressions of MHC ?,MHC? and CD86,promoted the infiltration of CD8+ T cells,up-regulated the mRNA expression of TNF-? and IFN-? and down-regulated TGF-?in tumor tissues.6.The molecular mechanism study of CS in CS adjuvant added DC vaccine showed that dectin-1 did not participate the CS induced DCs activation and TLR4 and TLR2 were CS receptors,of which TLR4 was the main receptor.The study has given us a deeper understanding of the immunoregulatory activity of CS,and is significant for sulfation modification related research.Besides,this study has provided the basis for immuno-adjuvant use of CS,and may open a new approach for hepatocellular carcinoma immunotherapy. |
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